Binuclear Ni^II‐DpaTyr Complex as a High Affinity Probe for an Oligo‐Aspartate Tag Tethered to Proteins

Abstract

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni(II)-DpaTyr (DpaTyr=bis((dipicolylamino)methyl)tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M=Zn(II), Ni(II), Mn(II), Cu(II), Cd(II), Co(III), and Fe(III)), we have found that Ni(II)-DpaTyr (1-2Ni(II)) displays a strong-binding affinity (apparent binding constant: K(app) approximately 10(5) M(-1)) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni(II) in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni(II)- and Zn(II)-DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni(II)-DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K(app)=2x10(9) M(-1)) was achieved between the Ni(II)-DpaTyr dimer 4-4Ni(II) and the D3x2 tag peptide (DDDNGDDD). This affinity is approximately 100-fold stronger than that observed in the binding pair of the Zn(II)-DpaTyr (4-4Zn(II)) and the D4x2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni(II)-DpaTyr probe and the D3x2 tag can also work effectively on a protein surface, that is, 4-4Ni(II) is strongly bound to the FKBP12 protein tethered with the D3x2 tag (DDDNGDDD) with a large K(app) value of 5x10(8) M(-1). Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tag-fused beta-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

“…Protein interactions with small ligands (including co‐factors and drugs) Protein/peptide interactions with metals and ions Protein/peptide interactions with nucleic acids …”

Real-Time Monitoring of ATP-Responsive Drug Release Using Mesoporous-Silica-Coated Multicolor Upconversion Nanoparticles

“…The size of the particle increased to 54.3 ± 3.1 nm ( n = 100) after coating with a mesoporous silica shell (Figure 1D,E, and Supporting Information, Figure S2). A tyrosine modified dipicolyl derivative TDPA was successfully grafted to the nanoparticle surface and used as a versatile precursor for chelating different metallic ions such as Zn 2+ and Cu 2+ to produce a metallic complex, 62 which further served as binding sites for oligo-aspartate and ATP (Figure S1). The successful grafting of binuclear metallic complex was confirmed using FTIR spectroscopy (Figure S3), UV–vis absorption spectroscopy (Figure 1F) and thermogravimetric analysis (Figure S4).…”

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

“…In addition to histidine‐mediated ion chelation, aspartate repeats can be applied for protein labeling as well. Several oligo‐aspartate‐tags are described (D 3 , D 4 , D 4x2 ) to fine tune the affinity of Zn 2+ ‐loaded probes, which have been successfully applied for N‐terminal labeling of the B 2 R and muscarinic acetylcholine receptor M1 using protocols, comparable to oligo‐His‐tag labeling [159–161] . Similar to the oligo‐His‐tags, oligo‐Asp‐mediated labeling is reversible due to the transient interaction with the metal ion, but can be converted into covalent POI modification by introducing a chloroalkane moiety into the metal loaded NTA and adding an N‐terminal cysteine residue into the oligo‐Asp‐tag [158] …”